1. Field of Invention
The present invention is in the field of recombinant DNA technology. This invention relates to a process for assembling multiple DNA fragments in vivo, and to the molecules employed and produced through this process. Thus, the method can be used for the rapid generation of recombinant constructs and for mapping phenotypically expressed mutations.
2. Description of Prior Art
Two of the fundamental tools of the field of recombinant DNA technology are the ability to recombine DNA, and the ability to localize (or map) the position of phenotypically expressed mutations.
Methods to assemble DNA fragments into plasmids that can replicate in vivo are of fundamental importance in the field of recombinant DNA technology. Such methods can be used, for example, to construct a plasmid bearing a particular gene being studied. Typically, such methodologies involve the introduction of a nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector (and the recovery of the amplified fragment). Examples of such methodologies are provided by Cohen et al. (U.S. Pat. No. 4,237,224), and Maniatis, T. et al., Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, 1982.
The desire to increase the utility and applicability of such methods is often frustrated by the lack of (suitable) restriction enzyme sites present at desired locations and, even when suitable restriction sites are present, by the methodological complexities involved in complex 3-way and 4-way ligations (such as sequential digestions, fragment isolation, and buffer incompatibility). Hence, it would be highly desirable to develop a general, simple, and rapid method to assemble multiple DNA fragments.
The polymerase chain reaction (PCR) technique was conceived and developed by the Cetus Corporation to provide for specific amplification of discrete fragments of DNA in order to allow simplified detection and purification of nucleic acid fragments initially present in a particular sample in only picogram quantities (Salki, et al. Science 230:1350-1354, 1985). The basic method is based on the repetition of three steps, all conducted in a successive fashion under controlled temperature conditions: (1) denaturing the double-stranded template DNA; (2) annealing the single-stranded primers to the complementary single-stranded regions on the template DNA; and, (3) synthesizing additional DNA along the templates by extension of the primer DNAs with DNA polymerase after 4 to 25 cycles of these steps; as much as a 100,000-fold increase in the amount of the original DNA is observed (Oste, BioTechniques 6:162-167, 1988). Reviews of the polymerase chain reaction are provided by Mullis, K. B. Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); and Mullis, K. B. et al. Meth. Enzymol. 155:335-350 (1987), which are incorporated herein by reference.
More recently, the PCR technology has been used for mutagenesis of specific DNA sequences and for other directed manipulations of DNA. For instance, PCR technology has been used to engineer hybrid (chimeric) genes without the need to use restriction enzymes in order to segment the gene prior to hybrid formation. In this approach, fragments that are to form the hybrid are generated in separate polymerase chain reactions. The primers used in these separate reactions are designed so that the ends of the different products of the separate reactions contain complementary sequences. When these separately produced PCR products are mixed, denatured, and reannealed, the strands having matching sequences at their 3'-ends overlap and act as primers for each other. Extension of this overlap by DNA polymerase produces a molecule in which the original sequences are spliced together to form a hybrid gene. Thus, this method requires four primers to construct a deleted, hybrid DNA molecule. Likewise, the method requires six primers and three rounds of PCR in order to construct a chimeric molecule (Horton, et al., Gene 77:61-68, 1989).
Recently Jones (U.S. Pat. No. 5,286,632) has described a method that can be used to join two DNA molecules. In this method, the polymerase chain reaction is utilized to add double-stranded regions to both ends of an insert DNA homologous to the ends of a linear vector. These homologous ends undergo recombination with a linear vector in vivo following transformation of Escherichia coli. This method can be used to introduce mutations into a preexisting vector but can not be used to rearrange (recombine) preexisting mutations present on separate DNA inserts. This method can be used to join at most two DNA molecules; this is a significant disadvantage since it is often desirable to join 3 DNA molecules. This method also requires that the homologous regions which undergo recombination be located at the ends of the DNA molecules amplified by PCR, thus necessitating the design and synthesis of primers unique for each particular pair of gene and cloning vector. An additional drawback of this method is a requirement to either purify both PCR fragments after amplification or to cut the template plasmids with a restriction enzyme that recognizes a site outside of the region to be amplified prior to amplification. An additional disadvantage of the in vivo cloning procedure described in the Jones patent (U.S. Pat. No. 5,286,632) is the extreme inefficiency of the recombination events; the recombination is less than one in 10,000,000-fold as efficient as transformation of intact plasmid.
Muhlrad et al. have described a method to introduce mutations into genes cloned into plasmids that replicate in yeast. (Muhlrad, D., Hunter, R., and Parker, R. Yeast 8:79-82, 1992). This method can not be used to rearrange (recombine) preexisting mutations present on DNA fragments. This method can be used to join at most 2 DNA molecules; this is a significant disadvantage.
Oliner et al. have described another method of in vivo cloning utilizing an E. coli strain with enhanced in vivo recombination (Oliner, J. D., Kinzler, K. W., and Vogelstein, B. Nucleic Acids Research 21:5192-5197, 1993). Although this method has an improved transformation efficiency compared to the methods described in the Jones patent (U.S. Pat. No. 5,286,632), the transformation efficiency is still approximately 100,000-fold lower than that of intact plasmid DNA transformed into E. coli, and the likelihood of efficient trimolecular and higher order recombinations would be extremely low.
Willem P. C. Stemmer (Stemmer, W. (1994) Nature 370, 389-391) has described a method for in vitro homologous recombination called DNA shuffling. In this method, pools of selected mutant genes are recombined in vitro by random fragmentation and PCR reassembly. In this method, the recombined molecules had to be cloned into an appropriate vector before transformation into E. coli and subsequent selection and analysis. This recloning step had to be repeated for each iteration of the selection process.
An alternative method for recombinational mapping of plasmid-borne genes in yeast has been described (Kunes, S., Ma, H., Overbye, K., Fox, M. S., & Botstein, D. (1987) Genetics 115: 73-81). The method of Kunes et al. relies upon the incidence of loss of a plasmid-borne mutation to identify its location. This method is based on a statistical analysis and provides a genetic map distance of a mutation from the end of a DNA fragment, the position of the end being determined by the fortuitous location of a restriction enzyme recognition site. This method is based on the loss of a plasmid-borne mutation to identify its location; in the process the mutation is lost rather than subcloned.
Ma et al. have presented a method for constructing plasmids in yeast by homologous recombination (Ma, H., Kunes,S. Schatz, P. J. and Botstein, D., Gene 58: 201-216). However, this method can not be used to rearrange (recombine) preexisting mutations present on DNA fragments. This method can be used to join at most 2 DNA molecules; this is a significant disadvantage.
Degryse et al. in vivo cloning by homologous recombination in yeast using a two-plasmid-based system Yeast 11,629-640. This method can be used to join at most 2 DNA molecules; this is a significant disadvantage. An additional drawback of this method is the requirement that one must first construct, using conventional methods, the two plasmids used in the two-plasmid-based system.